Collagen preparation for the controlled release of active substances

ABSTRACT

A collagen preparation for the controlled release of active substances is characterized in that it has mixtures of acid-insoluble collagens with different molecular weight distributions.

A number of polymers are used in the production of administration formsfor pharmaceutical or cosmetic active substances. Depending on the siteof application, these are used to impart to the respectiveadministration form the desired properties. Agents for the treatment ofwounds and implantable materials should be adapted to the surface of therespective application site, and they should not interfere with thefunction and activity of body cells, such as keratinocytes, fibroblasts,or endothelial cells. In these cases the spectrum of suitable polymersis therefore limited to those having an excellent compatibility oncontact with connective tissue and which preferably are biodegradable.

For quite a long time now, collagen, the main protein of the connectivetissue, has gained a special place among the polymers suitable for thispurpose. This is due to the biological compatibility and degradabilityof the administration forms produced therefrom. Such collagenpreparations are for the most part used as wound dressings without anyadditional additives. However, there have also been attempts to chargecollagen matrices of most different kinds with biologically activesubstances and to influence the release of these substances, which ingeneral follows the dissolution and/or enzymatic degradation of thecollagen carrier matrix, by the kind, structure and composition of thematrix.

To produce collagen matrices—such as those described in, for example,U.S. Pat. No. 5,024,841, U.S. Pat. No. 4,789,663, U.S. Pat. No.4,774,091, U.S. Pat. No. 4,563,350 U.S. Pat. No. 5,246,457, or EP 429438—solutions of telopeptide-free, acid-soluble collagen are used ingeneral. These are used to obtain reconstituted fibrils by means ofdialysis, shift of the pH, or other processes; and these fibrils arethen processed by means of different methods into preferably porousmatrices. These preparations can exhibit an excellent biocompatibilitysince they consist of pure collagen, but they have the disadvantage thata retardation of the active substance release is limited owing to theuse of soluble collagen.

Such a release retardation can be achieved by decreasing the solubilityof the collagen matrix by using cross-linking agents or binders, as isdescribed, for example in U.S. Pat. No. 4,409,332, DE 2 843 963, WO93/00890, or WO 85/04413, or by using coating agents, e.g., described inDE 38 41 397, or by combining water-soluble collagen with otherpolymers, preferably natural anionic polymers or their derivatives.However, owing to the additional components, additional toxicologicalrisks which, in particular when the known cross-linking agents forcollagen are used, should not be underestimated must be accepted in eachof these cases.

Another possibility of delaying the active substance release isdescribed in EP 518 697. Here, laminates are produced from water-solubleand/or water-insoluble collagen, which consist of one or several activesubstance-containing reservoir layers and one layer retarding the activesubstance supply. As compared to the above-mentioned preparations, aretardation of the release while minimizing the toxicological riskscould be achieved with such laminates. However, they have thedisadvantage that their production is extremely expensive and that theadherence of the layers can only be achieved with “moist” films. Dryfilms which are absolutely necessary in case of active substancessusceptible to decomposition cannot be joined to form such laminates,for example.

However, a retardation of the active substance release is not alwaysdesired. EP 224 453 describes a collagen matrix which mainly consists ofwater-insoluble, reticulated collagen and additionally compriseswater-soluble, non-reticulated collagen. In cosmetic preparations, e.g.,face packs, said soluble collagen serves as active substance which,after application, is dissolved out by means of the natural cutaneousmoisture or by extremely moistening the preparation which then takeseffect on the skin. For pharmaceutical purposes, active substances canbe incorporated into the preparation, these are then dissolved out andreleased from the collagen matrix together with the soluble collagenvery quickly after application. Such a collagen preparation can beuseful in cases where a rapid release is desired and appropriate.

Mechanisms to delay or accelerate the active substance release fromcollagen preparations have been described in many ways. However, due tothe given composition, each of these prior art collagen preparationsoffers only one possibility of influencing the release, and thereforethey have a given release profile each tailor-made to one specificproblem, e.g., a certain active substance or certain active substancegroup, a certain therapeutic principle, or a certain disease. None ofthe prior art collagen preparations can achieve a selective and at thesame time manifold control of the active substance release, i.e., permita variable and individual adaptation of the release kinetics of activesubstances to differing problems, wherein factors, such as differentactive substance properties and differences in the onset and duration ofaction, can be considered adequately for each case.

Accordingly, it was the object of the present invention of find acollagen preparation which is not only suitable for a certain activesubstance, a certain active substance combination, or a certain releaseprofile, but also permits—for a great variety of uses—a wide-ranging,reliable control of the active substance release adapted to therespective problem.

Most surprisingly, the solution of this object has been found in acollagen preparation for the controlled release of active substances,comprising mixtures of acid-insoluble collagens having differentmolecular weight distributions.

According to one embodiment, the collagen preparation comprisesdifferent active substances. It may additionally comprise adjuvants,such as viscosity regulators, binders, humectants, softening agents,penetration enhancers, preservatives, disinfectants, pH-regulators,antioxidants, active substance stabilizers, oils, fats, waxes, emulsionstabilizers, odorous substances, dyes, and/or inert fillers.

According to a preferred embodiment, the insoluble collagen istelopeptide-free, native, uncross-linked Type-1-collagen. This may be aninsoluble collagen which is a product obtained from calfskin by means ofalkaline decomposition.

Furthermore, the collagen preparation may be present in the form ofpowders, dusts, microparticles, fibers, flakes, foams, sponges, needles,small rods, tablets, gels, creams, single-layer films, or laminates.

Advantageously, the collagen preparation may include combinations ofdifferent administration forms to achieve the desired release kinetics.

It is preferred that the collagen preparation be bioadhesive.

A process for the production of the collagen preparation may comprisespray drying, freeze-drying, coating or casting with subsequent drying,phase separation and coacervation processes, compression, or fillinginto containers.

According to the process, the active substance release can additionallybe influenced and/or controlled by the mixing ratio of acid-insolublecollagens with different molecular weight distributions. According tothe process, the active substance release can additionally be controlledby dissolution, swelling, or erosion of the collagen preparation.Another possibility of controlling the active substance release is bybiological degradation of the collagen preparation.

The use of the collagen preparation according to the present inventionconsists in the controlled release of the active substance to wounds.However, the use may also be directed to the controlled release ofactive substances to intact skin. Finally, the use of the collagenpreparation may serve to implant or inject active substances into aliving organism.

In general, prior art collagen preparations for the active substancerelease are produced from acid-soluble collagen, that is collagen whichis dissolved clearly in dilute acids at a pH of 2. This acid-solublecollagen can be isolated from several animal and vegetable tissues bymeans of a great variety of processes.

In contrast to this, the collagen used according to the presentinvention is an acid-insoluble collagen which—when it is present inaqueous dispersion—cannot be brought into solution even whenconcentrated acetic acid is added.

It is preferred that this insoluble collagen be native collagen thegreater part of which Is present as Type-I-collagen and the smaller partas Type-III-collagen. The term native collagen refers to a collagenmolecule having an unchanged triple-helical tertiary structure.

The insoluble collagen used according to the present invention can beobtained from biomaterial of various origins by alkaline decomposition;for this purpose processes, such as those described, for example, inDE-OS 3034273, U.S. Pat. No. 4,021,522, or DE-OS 27 16 602 are slightlymodified. It is preferred that the starting material be calfskin, agedfor six months. In contrast to the normally used tissues and body partsof cattle, pigs, or horses, this ensures a starting material of adefined, constant quality, this in turn ensuring the reliablereproducibility of the process steps described in the following andillustrated in greater detail in Example 1 hereunder.

First, the calfskin is mechanically dehaired and degreased. Then,soluble collagens and non-collagenous soluble components of theconnective tissue are extracted and rejected. Next, the connectivetissue is first treated with an aqueous solution of an alkali hydroxide,preferably sodium hydroxide, and an alkali sulfate, preferably sodiumsulfate, and then with an aqueous alkali sulfate solution to saponifyand dissolve out sebaceous matter and to swell the collagen fibers in acontrolled manner. When this is done, the terminal, non-helical portionsof the collagen molecule, the so-called telopeptides which are mainlyresponsible for the antigenic properties of xenogeneic collagen, arealso split off. In addition, the treatment with alkali hydroxide/alkalisulfate solution and with pure alkali sulfate solution splits a definedportion of the intermolecular collagen bonds in the fiber composite ofthe swollen connective tissue, said portion depending on theconcentration and the duration of action of the solutions. Theintramolecular bonds of the collagen are not attacked so that thehelical structure of the molecule remains undamaged. The connectivetissue so decomposed is washed out with water and dilute acids inseveral stages, purified and neutralized, and then mechanicallycomminuted and dispersed in water.

The decisive step for the isolation of the acid-insoluble collagenhaving different molecular weight distributions used according to thepresent invention is the selective alkaline cleavage of theintermolecular collagen bonds. If, for example, an aqueous solution of9.75% sodium hydroxide and 9.2% sodium sulfate is allowed to react onthe swollen connective tissue for 48 hours, the HPLC-analysis (columnsystem Biosil TSK-400+Biosil TSK-125; eluent 0.5 m ammonium acetatebuffer pH 6.7; detector UV 275 nm; sensitivity range 0.02 AUFS) withassessment against standard-protein chromatograms shows an averagemolecular weight of about 420,000 for the insoluble collagen afterdispersion in water. In contrast to this, if the swollen connectivetissue is treated with an aqueous solution of only 5% sodium hydroxideand 9.2% sodium sulfate for only 12 hours, far fewer intermolecularbonds are split, and the analysis of the insoluble molecular aggregatesobtained after dispersion in water shows a mean molecular weight ofabout 2,500,000. These differences first of all affect the flow behaviorand with that the processibility of the dispersions of insolublecollagen. Whereas a dispersion with 5% of low-molecular, insolublecollagen is a viscous but still free flowing mass, the flow limit of adispersion of higher-molecular, insoluble collagen is at about 2.5%.

Thus, the variable factors of the decomposition process, by variation ofwhich different molecular weight distributions can be achieved eachtime, are concentration and reaction time of sodium hydroxide. Thehigher the concentration of sodium hydroxide and/or the longer itsduration of action, the lower the mean molecular weight of the isolated,acid-insoluble collagen, and vice versa. To represent the connectionbetween the molecular weight that can be achieved and the describeddecomposition conditions, e.g., in the form of a curve, one of theinfluencing factors, i.e., concentration or duration of action, wouldhave to be kept constant while the other one is changed. This ispossible in principle, but from the production and operational point ofview this is rather useless, since, for example, a free variation of theduration of action alone is not possible in general. For instance, verylong reaction times with a correspondingly low sodium hydroxideconcentration would increase both the machine running times and therequirement of personal; in view of the operating expenses this is notacceptable. On the other hand, owing to the required higher sodiumhydroxide concentration, very short durations of action result inincreased wear of the manufacturing facilities, e.g., of the conduitsand filtering installation, and this cannot be realized either becauseof increasing working expenses. Therefore, the required duration ofaction and concentration of sodium hydroxide must individually beascertained empirically for each problem in such as manner that theyrepresent an optimum both with respect to the demands on theadministration form, in particular regarding the release kinetics of theactive substance, and to economic efficiency.

The differences in the molecular weight distribution of insolublecollagen distinctly influence the properties of the collagenpreparations that are produced therefrom by means of drying. Example 2shows that, depending on the mixing ratio, foams of insoluble collagenwith different molecular weight distributions produced by means offreeze-drying have very different disintegration properties. While foamsconsisting of 100% of low-molecular, insoluble collagen completelydecompose in artificial wound exudation of pH 6.4 after only 45 minutes,foams made of 100% of higher-molecular, insoluble collagen have notdisintegrated even after 10 days under the same conditions.

The example shows that the use of high-molecular collagen aggregates hasa clearly stabilizing effect on the foam; this is expressed in a clearlyslowed down absorption of secretion and swelling, and in a very delayeddisintegration. Form stabilization by means of maintaining a highnatural cross-linking degree of the collagen has the importantadvantage, in particular from the toxicological point of view, that noadditional manipulations to consolidate the structure, e.g., by tanningor cross-linking, are required. The product is rendered dimensionallystable by merely maintaining the collagen's original quaternarystructure, such as that present in the skin, to a substantial degree.

Thus, the collagen foams, each manufactured from only one single basictype of insoluble collagen, show clear differences, for example, withrespect to interaction with the secretion of the wound. However, asmentioned above, the profile which is required for a collagen productand is based on a specific purpose can only in special cases besatisfied by such mono-preparations. Compared with that, the presentinvention offers two possibilities of exactly tailoring the propertiesof a collagen preparation to given demands on a product. On the onehand, the molecular weight distribution of the insoluble collagen cancontinuously be varied over a very wide range by selectively controllingthe cleavage of the intermolecular bonds. On the other hand, therequired product properties can be adjusted by mixing said variations ofinsoluble collagen each having different molecular weight distributions.If the already mentioned Example 2 is continued, this becomes clear, forinstance, by the disintegration of freeze-dried foams, where—prior todrying—insoluble collagen having a mean molecular weight of about420,000 and insoluble collagen having a mean molecular weight of about2,500,000 were mixed at always different ratios. With differentpH-values, the decay periods always show a gradual decrease when thepercentage of insoluble collagen having a low average molecular weightis gradually increased.

The exact adjustment of the product properties to a demand profilerepresenting the optimum for the respective therapy is of importanceprimarily in the therapy using pharmaceutical active substances.

Since the collagen preparation according to the present invention ispreferably used on the skin, on external and internal wounds, and ininternal body tissues and body cavities after implantation or injection,the active substances suitable for charging the collagen preparationpreferably are active substances for the dermal and transdermalapplication, active substances for the treatment of wounds and for thepromotion of wound-healing, as well as active substances usuallyadministered by means of preparations for implantation or injection.

For the dermal treatment of local skin diseases the following substancesare used: local anaesthetics, local antibiotics, antiseptics,antimycotics, antihistaminics, and antipruritic drugs; keratolytics andcaustic drugs; virustatics, antiscabietic agents, steroids, as well asdifferent substances for the treatment of acne, psoriasis, orphotodermatoses. Active substances applied intradermally include, forexample, steroid and non-steroid antirheumatics, substances stimulatingthe blood flow, or vasoprotectors and vaso constrictors for thetreatment of vascular diseases. The active substances appliedtransdermally include, for example, neuroleptics, antidepressants,tranquilizers, hypnotics, psychostimulants, analgesics, musclerelaxants, antiparkinson drugs, ganglionic blockers, sympathomimetics,alpha-sympatholytics, beta-sympatholytics, antisympathotonics,antidiabetics, coronary therapeutic agents, antihypertensives,anti-asthmatics, or diuretics. The collagen preparation according to thepresent invention can also be used on the skin in cosmetic preparations,e.g., in the form of lather masks or films for the treatment of, forexample, aged skin, wrinkles, or impure skin; for body care, depilation,reduction of perspiration, or for light protection.

Active substances which are used in the collagen preparations accordingto the present invention on external and internal wounds, preferably arestyptic active substances, among which collagen itself has a specialplace; wound-cleansing substances, such as enzymes, antiseptics,disinfectants, and antibiotics; as well as active substances promotingwound healing which stimulate granulation, induce vascularization, orpromote epithelization. Among the active substances promoting the woundhealing, biologically active peptides and proteins—which develop highactivities at only very low concentrations and are mainly manufacturedby recombinant technologies—increasingly gain importance. The collagenpreparation according to the present invention represents a particularlysuitable carrier and release system for these substances which includethe so-called growth factors, such as Platelet derived growth factor(PDGF), Epidermal growth factor (EGF), Platelet derived endothelial cellgrowth factor (PD-ECGF), acidic Fibroblast growth factor (aFGF), basicFibroblast growth factor (bFGF), Transforming growth factor α (TGF α),Transforming growth factor β (TGF β), Keratinocyte growth factor (KGF),Insulin-like growth factors 1 and 2 (IGF1, IGF2), and Tumor necrosisfactor (TNF).

The active substances administered parenterally by means of the collagenpreparation according to the present invention include, for example,antibiotics, antiseptics, anaesthetics, analgesics of varying strengths;cytostatics, hormones, steroids, cytokinins, such as interleukins,interferons, and colony-stimulating factors, Hormone releasing andrelease inhibiting factors, prostaglandins, enzymes, as well as growthfactors, in particular osteoinductively effective bony growth factors.

One of the greatest challenges for the development of activesubstance-containing preparations is to find formulations releasing theactive substance in such a manner that the optimum action and besttherapy is achieved. The half-life period of many active substances, inparticular of the above-mentioned biologically active peptides andproteins, in the body is relatively short, and for this reason they mustfrequently be administered several times a day.

Therefore, active substance vehicles releasing the active substances ina delayed or even controlled manner gain increasing importance. Collagenpreparations according to the present invention offer the possibility ofdeveloping carrier systems using relatively few formulation basecomponents. These systems are very flexible both with regard toapplication and design, and can exactly be directed to the requiredsolution of a problem. The mechanisms, which impart to the releasekinetics of an active substance its characteristic features, canselectively be controlled by mixing of insoluble collagen havingdifferent molecular weights and by shaping of the collagen preparation.These factors, in addition to structure and density of the polymericcollagen skeleton, also influence the number and distribution ofhydrophilic, hydrophobic and ionic bonds along the polymer skeleton.Since active substance can not only be enclosed in cavities of acollagen preparation according to the present invention, but can also beadsorbed to the surface of the collagen skeleton, the bonding forcebetween collagen and active substance—and thus its release behavior—issubstantially-determined by ionic relations as well as by hydrophilicand hydrophobic interactions.

In the dermal, intradermal and transdermal application of an activesubstance using a collagen preparation according to the presentinvention, the solubility of the active substance in the preparation,the degree of charge and saturation, and the diffusion rate of activesubstance within the preparation—in addition to structure, density andbonding activity of the collagen preparation—have an influence on therelease behavior.

In case of collagen preparations for the release of active substances toexternal or internal wounds and collagen preparations for implantationor injection another factor appears.

Since the collagen preparations according to the present invention comeinto contact with body fluid in the applications mentioned above, therate and amount of liquid absorbed by the collagen preparation andconsequently the swelling capacity and disintegration properties of thecollagen preparation can be used to control the release, as is shown inExample 2 mentioned hereinbefore. In addition to the decay of thecollagen preparation used as controlling mechanism, other suitablemechanisms with regard to the release control on contact with body fluidprimarily include the dissolution of active substance out of thecollagen preparation by means of body fluid and the fluid-induceddiffusion of active substance from the center of the collagenpreparation to its interface. Furthermore, the release is influenced bythe biological degradation of the collagen preparation by means ofhydrolysis and enzymatic reaction on contact with body fluid. The widerange of interaction between the liquid and preparations of mixtures ofinsoluble collagen having different molecular weight distributions hasalready been shown in Example 2. The way said interactions incombination with one or several of the above-mentioned influencingfactors take effect on the release kinetics of an active substance isillustrated Examples 3 and 4. It is made clear that the mixing ratio ofinsoluble collagen having different molecular weight distributionssignificantly influences the kinetics of the active substance release.In the case of a given active substance and a given desired release pertime unit, it is thus possible to adapt the collagen preparationselectively to the required demands; in this way an optimum activesubstance dosage for the respective therapy is achieved afterapplication over the pre-determined application period. If activesubstance is to be released over an application period of 24 to 48 hoursfrom the administration form chosen in Examples 3 and 4—manufactured ofthe preparations of insoluble collagen having different molecular weightdistributions as described in Example 1—the mixing ratio oflow-molecular and high-molecular insoluble collagen should not be lowerthan 3 to 1. If, on the other hand, an even, delaying active substancerelease from a comparable administration form is to take place over anapplication period of 7 to 14 days, the mixing ratio of low-molecularand high-molecular insoluble collagen should not be greater than 1 to 3.

In order to prove the controlling possibilities of preparationsconsisting of mixtures of insoluble collagen having different molecularweight distributions, free from any influence of other auxiliary agents,pure collagen preparations plus active substance were exclusively usedin the Examples on purpose. In practice, however, it will not bepossible to do without additional water-soluble or water-dispersibleadditives, since, in addition to the requirements regarding the activesubstance release, the intended purpose will usually also result indemands with respect to handling properties and stability of an activesubstance-collagen-preparation.

Such adjuvants may be additional polymers, e.g., serving as viscosityregulators when liquid preparations are used or as binders when solidforms are used, for example, cellulose derivatives, starch derivatives,galactomannan derivatives, dextrans; vegetable polysaccharides, such asalginates, pectins, carrageenan, or xanthan, chitosan, proteins,glycoproteins, proteoglycans, glucosaminoglycans, polyvinyl alcohol,polyvinylpyrrolidone, vinyl pyrrolidone-vinyl acetate copolymers,polyethylene glycol, polyacrylates and polymethacrylates, polylactidesand polyglycolides, as well as polyamino acids.

The collagen preparation may comprise as additional adjuvants:

humectants, such as glycerol, sorbitol, polyethylene glycol,polypropylene glycol,

softening agents, such as citric acid ester, tartaric acid ester, orglycerol ester,

penetration enhancers, such as alkyl sulfates, alkyl sulfonates, alkalisoaps, fatty acid salts of multivalent metals, betaines, amine oxides,fatty acid esters, mono-, di- or triglycerides, long-chain alcohols,sulfoxides, nicotinic acid ester, salicylic acid, N-methyl pyrrolidone,2-pyrrolidone, or urea,

preservatives, such as p-Cl-m-cresol, phenylethyl alcohol, phenoxyethylalcohol, chlorobutanol, 4-hydroxybenzoic acid methylester,4-hydroxybenzoic acid propylester, benzalkonium chloride,cetylpyridinium chloride, chlorohexidine diacetate or digluconate,ethanol, or propylene glycol.

Disinfectants, for example, halogens, such as polyvidoneiodine; halogencompounds, such as sodium hypochloride or tosylchloride sodium;oxidants, such as hydrogen peroxide or potassium permanganate; arylmercury compounds, such as phenylmercury borate or merbromin; alkylmercury compounds, such as thiomersal; organotin compounds, such astri-n-butyltin benzoate; silverwhite compounds, such as silverwhiteacetyltannate; alcohols, such as ethanol, n-propanol, or isopropanol;phenols, such as thymol, o-phenylphenol, 2-benzyl-4 -chlorophenol,hexachlorophene, or hexylresorcinol; or organic nitrogen compounds, suchas 8-hydroxyquinoline, chloroquinaldol, clioquinol, ethacridine,hexetidine, chlorohexidine, or ambazone.

pH-regulators, such as glycerol buffers, citrate buffers, boratebuffers, phosphate buffers, or citric acid phosphate buffers.

Antioxidants, such as ascorbic acid, ascorbyl palmitate, tocopherolacetate, propyl gallate, butylhydroxyanisole, or butylatedhydroxytoluene.

Active substance stabilizers, such as mannitol, glucose, lactose,fructose, saccharose,

emulsifyable inactive ingredients, such as oils, fats, and waxes,

odorous substances, dyes, cleaning agents, substances for personalhygiene,

emulsion stabilizers, such as non-ionogenic emulsifiers, amphotericemulsifiers, cation-active emulsifiers, and anion-active emulsifiers,

fillers, such as micro-crystalline cellulose, aluminum oxide, zincoxide, titanium oxide, talcum, silicon dioxide, magnesium silicate,magnesium aluminum silicate, kaolin, hydrophobic starch, calciumstearate, or calcium phosphate.

For example, adjuvants may be dissolved, dispersed, or emulsified indispersions of insoluble collagen prior to shaping the administrationform. However, they may also be introduced in a manner usual and knownfor the respective shaping processes after termination of a primaryforming process (described hereinafter) of the mixtures of insolublecollagen during later stages of the drug shaping.

The embodiments wherein admixtures of insoluble collagen of differentmolecular weight distributions may be used for the controlled release ofactive substances are so numerous and manifold that they cannot berepresented herein extensively.

These embodiments of the collagen preparation according to the presentinvention may be manufactured from mixtures of dispersions of insolublecollagen each having different molecular weight distributions accordingto various methods known to the skilled artisan; for example by spraydrying, freeze-drying, coating or casting with subsequent drying, phaseseparation and coacervation processes for particles or emulsifieddroplets, drying and compression, as well as by simple filling intocontainers, e.g., tubes. They result in, for example, powders, dusts,microparticles, fibers, flakes, foams, sponges, needles, small rods,tablets, gels, creams, single-layer films, or laminates. Preferredembodiments are spraydried microparticles, freeze-dried foams, andgel-like films manufactured by coating.

The introduction of active substance into the collagen preparationaccording to the present invention may be effected such that the activesubstance is dissolved or dispersed in the finished mixture ofdispersions of insoluble collagen having different molecular weightdistributions, prior to forming the preparation. If more than one activesubstance is to be incorporated, these may also be dissolved ordispersed separately from one other in the individual fractions ofinsoluble collagen, prior to the mixing process. Active substance mayalso be introduced on or into the preparation after the shaping processof the preparation by means of coating, spraying, impregnating, dipping,or other adsorption methods.

The possible embodiments, the respective manufacturing processes, andthe methods of incorporating the active substance may also be combinedwith one another in order to achieve certain properties.

For instance, the collagen preparation according to the presentinvention may comprise a shell of insoluble collagen with a low meanmolecular weight, which is soluble or at least swellable in body fluid,in the form of a sponge or film containing a micro-particulatepreparation of insoluble collagen having a high mean molecular weightdispersed therein. If only one active substance is incorporated, theouter sponge or film phase serves the quick release of active substanceto achieve the required minimum active substance concentration veryquickly, followed by a slower, more even release of active substancefrom the inner, particulate phase to maintain the required activesubstance concentration over the application period. Such releaseprofiles may also be achieved with other preparation forms, such asfibers in hydrogels, spongy oil-in-water-emulsions, compressed mixturesfor implantable tablets, multi-layer films, combinations of films andfoams, and the like.

Such multi-phase preparations can also be used if a release of differentactive substances at different points of time with different releaserates is desired, or if one or several active substances are to bereleased phase-like, i.e., with release-free intervals. In this caseactive substance A may, for example, be contained in a readily soluble,quick-releasing outer phase, whereas active substance B is contained inan inner phase which is slightly soluble and releases the activesubstances in a retarded and controlled sustained manner. If aphase-like release profile is desired for only one active substance,external and internal phase may, for example, release active substanceat an even kinetics. However, the inner phase will then be surrounded byan active substance-free layer of insoluble collagen, which, afterdissolution of the outer phase or active-substance-exhaustion thereof,must swell or dissolve itself first so that the active substance releasefrom the inner phase can take place. By means of this arrangement, aninterval without any active substance release can be obtained.

If the collagen preparation according to the present invention is usedfor the dermal, intradermal or transdermal application of pharmaceuticalactive substances or cosmetic active principles, plane embodiments, suchas films, membranes, or thin sponges are preferred. These flatembodiments may consist of laminates which, for example, also includebarrier layers which are free from active substances, permeableseparating layers, controlling membranes, and adhesive layers.Substructures, such as laminae, powders, microparticles, or oildroplets, may then be introduced in or between the individual layers, inorder to achieve the suitable release kinetics for one or several activesubstances. It is preferred that such single- or multi-layer collagenpreparations for the mentioned applications, e.g., for protectionagainst dehydration or growing in of germs, be provided with a backinglayer and a removable protective layer located on the opposite sideaccording to known processes, the backing layer and the protective layerconsisting of materials known to the skilled artisan, for example, thoseused in the formulation of patches and adhesive tapes.

In a preferred embodiment of the collagen preparation according to thepresent invention for the use on external wounds and in the interior ofthe body, the preparation is paste-like, e.g., foamy or spongy. The sizeof the pores and the structure of the preparation are designed such thatimmigration of cells, e.g., fibroblasts or osteoblasts, into thepreparation is possible and that the cells are given a structuralorientation; this can particularly be attributed to the degree oforientation of the collagen in the preparation according to the presentinvention, which is similar to that of natural connective tissue.Immigration of the cells may be necessary, for example, for thedegradation of the preparation or for the release or deposition ofsubstances which are required, for example, for neo-formation of tissueor vascularization of a tissue which is to take the place of thecollagen preparation according to the present invention.

In another preferred embodiment for the use on wounds or in the interiorof the body, the collagen preparation according to the present inventionis adjusted by admixing adjuvants, such as carboxymethylcellulose,polyacrylic acid, tragacanth, sodium alginate, or hydroxypropylcellulosein such a manner that it is bioadhesive, i.e., that it adheres to thesurface tissue of the application site for a certain time by means ofinterfacial forces, in order to increase the retention time at the siteof application or absorption.

EXAMPLES

1. Extraction of Insoluble Collagen from Calfskin

1.1 Treatment with Ca(OH)₂ 1% in H₂O 100 h Treatment with NH₄Cl 3% inH₂O 1 h Treatment with NaOH 5% + Na₂ SO₄ 9.2% in H₂O 12 h Treatment withNa₂ SO₄ 1 molar in H₂O 0.5 h Treatment with HCl 1% in H₂O 1.5 hTreatment with H₂O₂ 0.5% in H₂O 6 h

After mechanical comminution and dispersion in H₂O (pH 6.0) theinsoluble collagen has an average molecular weight of about 2,500,000.The collagen concentration amounts to 0.75%.

1.2 As in a), difference:

Treatment with Ca(OH)₂ 1% in H₂O 72 h Treatment with NaOH 9.75% + Na₂SO₄ 9.2% in H₂O 48 h

After mechanical comminution and dispersion in H₂O (pH 6.0) theinsoluble collagen has an average molecular weight of about 420,000. Thecollagen concentration amounts to 0.75%.

2. Production of Active Substance-free Lyophilizates from Mixtures ofthe Dispersions According to 1.1 and 1.2 and Determination of theDisintegration Time in Buffer Mixtures having Different pH-values.

The following mixtures of the dispersions according to 1.1 and 1.2 wereproduced:

Dispersion 1.1 Dispersion 1.2 A 100%   0% B 75% 25% C 50% 50% D 25% 75%E  0% 100% 

24 g of the mixtures A to E were each filled into deep-drawing dies of6×4×1.5 cm, shock-frozen and brought to a temperature of −50° C. within60 minutes. Subsequently the mixtures were freeze-dried.

The disintegration time of the resulting sponges (dimensions 6×4×0.8 cm)was determined under slight stirring in 100 ml of the following buffermixtures:

pH 3: buffer mixture citric acid/sodium chloride/sodium hydroxidesolution with addition of a fungicide

pH 5: acetic acid (0.1 molar) sodium acetate (0.2 molar)

pH 6.4: artificial exudation of a wound (without albumin)

pH 7.5: potassium hydrogenphosphate/NaOH (0.1 molar/0.1 molar)

Results:

pH of buffer solution Mixture 3 5 6.4 7.5 A 2 h 15 min >10 d >10 d >10 dB 1 h 10 min 3 d 3 d 2 d C 1 h 24 h 24 h 6 h D 20 min 4 h 10 min 4 h 30min 1 h 45 min E 2 min 35 min 45 min 15 min

3. Production of Lyophilizates with p-Hydroxybenzoic Acid Propylesterand Determination of the Release

0.275% (relative to the dispersion) of p-hydroxybenzoic acid propylesterwere dissolved in each of the mixtures A to E according to Example 2. 10g of each of the mixtures were filled into deep-drawing dies accordingto Example 2, shock-frozen, cooled to −50° C. within 60 mins., and thenfreeze-dried.

About 30 mg (theoretical PHB-ester-content 6.1 mg) of each of theresulting lyophilizates (sponge weights about 135 mg, theoreticalPHB-ester-content 27.5 mg) were placed in a paddle-device and stirred at45 rpm in 500 ml of 0.05 N sodium hydroxide solution at 37° C. After 4hours, 10 ml of each release medium were withdrawn. The active substancecontent was determined by ultraviolet-photometry against standard at 294nm at a layer thickness of 1 cm.

Results:

Mixture A B C D E released act. subst. (mg) 2.1 2.7 3.4 3.8 * * couldnot be determined because of complete disintegration of lyophilizate

4. Production of Lyophilizates with Lidocaine Hydrochloride andDetermination of the Release

0.042% (relative to the dispersion) of lidocaine hydrochloride weredissolved in each of mixtures A to E according to Example 2. 24 g ofeach mixture were filled into deep-drawing dies according to Example 2,shock-frozen, cooled to −50° C. within 60 minutes, and thenfreeze-dried.

The resulting lyophilizates (sponge weights about 250 mg, theoreticalcontent of lidocaine hydrochloride 10 mg) were each filled into apaddle-device and fixed at the bottom of the vessel by means of a net.350 ml of water having a temperature of 37° C. were filled into thedevice and stirred at 45 rpm. After 24 hours, samples of the releasemedium were taken. The active substance content was determined at 262.5nm and a layer thickness of 1 cm by means of ultraviolet-photometry andassessment based on a standard curve.

Results:

Mixture Active substance released A 1.2 mg B 5.1 mg C 7.4 mg D 8.8 mg E10.0 mg 

What is claimed is:
 1. A collagen preparation for the controlled releaseof active substance which comprises at least one active substance and amixture of acid-insoluble collagen-fractions each fraction having adifferent mean molecular weight and said fractions being obtained byalkaline decomposition.
 2. A collagen preparation according to claim 1,wherein the collagen preparation comprises a plurality of activesubstances.
 3. A collagen preparation according to claim 1, comprisingan adjuvant selected from the group consisting of viscosity, regulators,binders, humectants, softening agents, penetration enhancers,preservatives, disinfectants, pH-regulators, antioxidants, activesubstance stabilizers, oils, fats, waxes, emulsion stabilizers, odoroussubstances, dyes, inert fillers and mixtures thereof.
 4. A collagenpreparation according to claim 1, wherein the insoluble collagen istelopeptide-free, native, uncross-linked Type-1-collagen.
 5. A collagenpreparation according to claim 4, wherein the insoluble collagen is aproduct obtained from calfskin by alkaline decomposition.
 6. A collagenpreparation according to claim 1, in the form of powders, dusts,microparticles, fibers, flakes, foams, sponges, needles, rods, tablets,gels, creams, single-layer films, or laminates.
 7. A collagenpreparation according to claim 6, which comprises a combination ofdifferent forms of the collagen in order to obtain a desired kinetics ofactive substance release.
 8. A collagen preparation according to claim1, which is bioadhesive.
 9. A process for the preparation of a collagenpreparation according to claim 1, which comprises combining at least oneactive substance with a mixture of acid-insoluble collagen-fractions andsubjecting such combination to spray drying, freeze-drying, coating orcasting with subsequent drying, phase separation and coacervationprocesses, compression, or filling into containers.
 10. A processaccording to claim 9, wherein the active substance release is controlledby the mixing ratio of acid-insoluble collagens having different meanmolecular to weights.
 11. A process according to claim 9, wherein theactive substance release is controlled by dissolution or swelling andsubsequent erosion of the collagen preparation.
 12. A process accordingto claim 9, wherein the active substance release is controlled by thebiodegradation of the collagen preparation.
 13. A method for thetreatment of a wound which comprises applying to the wound a collagenpreparation as defined in claim
 1. 14. A method for the use of acollagen preparation as defined in claim 1, which comprises applyingsaid collagen preparation to intact skin.
 15. A method for the use of acollagen preparation as defined in claim 1, which comprises implantingor injecting said collagen preparation into a living organism.